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Immunoelectrophoresis of the proteins of the corneal epithelium
/'2.1q~. Eye. Rc.v. (L963)2, L96-20:]
lmmunoelectrophoresis of the Proteins of the Corneal Epithelium J U L E S .~ [
O'phthalmolog'icat Clinic, of the University of Ghe.nt, Belgium (Receiced 27 Febr~lary 1962 and in ret,'isedJbrm 12 Ju~e 1962) The protehls of the corne~l epithelium are separated into a greater number of tractions by high-tension agar electrophoresis than by paper electrophoresis. The distribut i o n o f t h e d i f f e r e n t fr,~ctions is g r e a t l y i n f l u e n c e d b y t h e i o n i c s t r e n g t h o f t h e b u f f e r s o l u t i o n . A l t h o u g h in a g a r e l e e t r o p h o r e s i s a l a r g e r n u m b e r o f f r a c t i o n s a r e f o u n d , t h e r e is, n e v e ~ h e l e s s , a g r e a t s i m i l a r i t y b e t w e e n t h e p a t t e r n o b t a i n e d a f t e r p a t ) e r e l e c t r o phoresis and that obtained after agar electrophoresis at relatively high ionic strength. T h e e x i s t e n c e o f a g r e a t e r n u m b e r o f f r a c t i o n s is c o n f i r m e d b y i m m u n o e l e c t r o p h o r e s i s , which reveals at least eight different precipitation liues corresponding to different tissue proteins. T h e f r a c t i o n p r e s e n t i n h i g h e s t c o n c e n t r a t i o n , w h i c h wo h a v e c a l l e d t h e p r i n c i p a l f r a c t i o n , is f o u n d n o t o n l y i n h u m a n s , b u t a l s o in s e v e r a l o t h e r m a m m a l s . I n v i e w o f t h e f a c t t h a t , in all c a s e s , t h e c o r r e s p o n d i n g p r e c i p i t a t i o n l i n e a p p e a r e d w h e n a n t i s e r u m to bovine corneal epithelium was used, it would seem that the principal fraction has a v e r y d e f i n i t e o r g a n s p e c i f i c i t y . T h i s f r a c t i o n h a s p o o r s t a b i l i t y in s o l u t i o n . I t r a p i d l y decomposes into two or three components, which are separated during electrophoresis in agar, where their mobiHties are slightly different.
1. Introduction
Several authors have studied the water-soluble proteins of the corneal epithelium b y paper electrophoresis (Franpois and l%abaey, 195~, 1956; Miinich and Oswald, 1955; Takak~u, 1957; Steinvorth, 1958, 1959a,b, 1960a,b). Other authors have studied the soluble proteins of the entire cornea, h~cluding the epithelium and the parenchyana, TA]~LE I
Soluble prolein f~'action.s' of bovine corneal e'oithelium after separation by paper electro phorcsis Pi-otcin freer ion 1
l"ranqois a n d R a b a e y ( 1954 )
)litnich a n d Oswald ( 1 !)55)
Steinvorth (1960a)
l(~awerau a n d O t t ( 1901 )
12
3-63
19"3
ll'0
]I ]] I
(13 O
20-93 59-55
60"5 12 "0
05-0 16 "3
j \r
7
15-80
8-2
3.7
4.0 ~:a]tles
aT'~,
o., ,.o total lwoteins.
e.g. Guidry, Kelly and Allen (1956), who resorted to the determination of the sedi m e n t a t i o n constants after ultracentrifugation. Recently, l ( a w e r a u and Oft (1961) m a d e a st u d y of the soluble proteins of the three structural layers of the cornea, the epithelium, the substantia propria and the endothelium, also by paper eleetrophoresis. Most of these investigators have distinguished av least four soluble proteins in the paper electrophoretic p a t t e r n of the bovine corneal epithelium, b u t t h e y have reported different figures for the distribution of these fractions, as shown in Table I. Fraction III, which shows P~kS-positivity on paper electrophoresis (Franpois and 196
1MM. I J N O I g L E Q ' I Y I . I O P I t O I I , I g S I N O F C O I / N I ~ A I ,
EPI'I?I~I']I,1UM
197
I~abaey, 1954, ] 956), 1)robably c o n t a i n s g l y c o g e n in c o m p l e x w i t h proteiJls (Frangois, i~almey mxd W e i n s t e i n , 1_961). T h e s a m e a u t h o r s also f o n n d t h a t on t h e p a p e r electrop h e r o g r a m ~ o n l y ti'aetion IJ s t a i n e d s l i g h t l y w i t h S u d a n 131acl¢ 13, s u g g e s t i n g t h e presence of o n l y a small q u a r t t i t y of lipoproteiJm (FrangoLs a~ld ]Labaey, 1954, 1956). S t e i n v o r t h (1958), h o w e v e r , f o u n d t w o l i p o p r o t e i n tYactions w h i c h , air.hough iJ~ different p r o p o r t i o n , b o t h s t a i n e d p o s i t i v e l y for p h o s p h o l i p o p r o t e h l s . The agar electrophoretic and imarmnoeleetrophoretic studies presented in this p a p e r were c a r r i e d o u t for t h e p u r p o s e of c h a r a c t e r i z i n g m o r e a c c u r a t e l y t h e d i f f e r e n t w a t e r - s o l u b l e p r o t e i n s of t h e c o r n e a l e p i t h e l i u m , t h e n a t u r e a n d f u n c t i o n of wlfich are still a l m o s t c o m p l e t e l y u n k n o w n . 2. M a t e r i a l s and M e t h o d s
The bovine a n d other a n i m a l eyes were o b t a i n e d within 10 min after the d e a t h of t h e animal. The h u m a n eye was o b t a i n e d a t operation, the ey.e being removed for uveal melanoma. The corneal and c o n j u n c t i v a l surfaces of the eyes were wiped w i t h a cloth soaked in l~inger's solution to remove t h e blood. The epithelial cells of the cornea were isolated w i t h a Graefe knife b y a t e c h n i q u e w h i c h has been previously described (lq'ran~ois and R a b a e y , 1954, ]956). I n general, it is relatively easy to separate the entire epithelium from t h e u n d e r l y i n g stroma. :For i m m u n i z a t i o n , t h e bovine corneal epithelium was homogenized in :Ringer's solution, using 100 mg of fresh tissue per ml of solution. The suspension was t h e n centrifuged a t 20,000 × g for 20 rain. One milliliter of the s u p e r n a t a n t , which c o n t a i n e d a b o u t 1 % proteins (protein concent r a t i o n d e t e r m i n e d b y t h e B i u r e t method), was injected once each week for 6-8 weeks s u b c u t a n e o u s l y into r a b b i t s weighing a b o u t 2 kg. The serum of t h e i n j e c t e d animals was removed 2 weeks after the l a s t injection. A certain a m o u n t of this a n t i s e r u m was absorbed with the n o r m a l bovine serum, as described b y ]3urtin (1960). U p to 0"1 ml of bovine serum was a d d e d to 1 ml of antiserum. D u r i n g immunodiffusion or immunoelectrophoresis, no p r e c i p i t a t i o n lines occurred, w h e n t h e bovine serum was developed w i t h t h e absorbed antiserum. :For electrophoresis, the corneal epithelium was homogenized in R i n g e r ' s solution or in distilled w a t e r using 200 m g of fresh tissue per m | of solution. After c e n t r i f u g a t i o n as indicated above, the clear s u p e r n a t a n t , which c o n t a i n e d a b o u t 2 % protein, was used for electrophoresis a n d immmmelectrophoresis w i t h o u t dialysis. On storage, a g r a d u a l clouding was observed in the solution and, after a few hours, a slight precipitate was formed. Electrophoresis was carried o u t in agar on microscopic slides a t 4°C a n d a t h i g h tension (20 V/cm) (Rabaey, 1959; Wieme, 1959a,b). C o n s t a n t t e m p e r a t u r e was achieved b y means of an a u t o m a t i c cooling s y s t e m in which p e t r o l e u m ether (b.p. 30°-50°C) was used in t h e electrophoresis t a n k as i m m e r s i n g fluid. As s u p p o r t i n g medium, either Difco Special Ag~r Noble* (1~/o in veronal buffer, p H 8-~, _P/2 0.05) or Difco Bacto-Agar* (1~/o in veronal buffer, ptff 8-4, I / 2 0-025) was used. On completion of electrophoresis (25 rain), the a g a r plates were fixed in 6 5 % e t h y l alcohol c o n t a i n i n g 5~/o acetic acid (w/w) for 1-24- hr. After d r y i n g u n d e r filter paper, t h e plates were finally stained a n d scanned. The s t a i n i n g solution was composed of 0"5 g of amido black 10B, 40 ml of normal acetic acid, 10 m l of normal sodium acetate, 10 m l of glycerin a n d 40 ml of distilled water. ~tfter s t a i n i n g for 30 rain, t h e agar plates are differentiated in 2 % acetic acid. Scanning was performed a t 546 m~x in a modified :Eppendorf p h o t o m e t e r (\Vieme, 1959a). The density m~rves were o b t a i n e d b y p l o t t i n g the absorbance A of the s t a i n e d protein fractions a g a i n s t the relative m o b i l i t y m~. The relative m o b i l i t y ~ of the different fractions was d e t e r m i n e d as described earlier ( R a b a e y a n d Verriest, 1957), using d e x t r a n (mr=O) a n d h u m a n serum a l b u m i n ( m r s 1 ) as t e s t substances. * Dffco : L a b o r a t o r i e s , D e t r o i t , Mich.
198
,JULES
FRANq~OIS
AND
MAgC.~]L
I~ABAEY
I m m u n o e l e c t r o p h o r c s i s was also carried out on microscopic slides as suggested by the Scheidcgger technique (1955). A trough, 1 m m wide a n d 6-7 cm long, was m~,.de in the middle of the agar plate, r u n n i n g parallel to the long side of the slide. The e x t r a c t of the corneal epithelium was deposited in the centre of the plate, a b o u t 3 m m from both sides of t h e trough in a slot 1-2 m m long. Electrophoresis was t h e n carried o u t as described above, after which t h e trough was filled with a n t i s e r u m and the plates were allowed to d e v e l o p for 24 hr. The best results were obtained with u n d i l u t e d antiserum. l m m u n o d i f f u s i o n tests (double-diflhsion t e c h n i q u e according to Ouchterlony, 1958) were carried out on micro-plates usi~g two different templates (Fig. 6). T h e first t e m p l a t e consists of a central well, filled w i t h antiserum, stu-rounded b y several others filled with different antigens; in t h e second t e m p l a t e the different antigen wells are situated along a trench-like well filled with antiserum. 3. R e s u l t s
Agar electro~horetic analysis
T h e d e n s i t y ctu'ves of a g a r e l e c t r o p h e r o g r a m s o b t a i n e d w i t h proteh~s f r o m b o v i n e c o r n e a l e p i t h e l i l m l a t t w o d i f f e r e n t ionic s t r e n g t h s are s h o ~ m in :Fig. 1 (Difco Special A g a r ~ o b l e , 1"/2 0.05) a n d Fig. 2 (Difco B a c t o - A g a r , /"/2 0-025). I t is o b v i o u s t h a t t h e p a t t e r n s o b t a i n e d w i t h t h i s m e t h o d are m o r e c o m p l e x th,~n those obtained b y paper elcctrophoresis. N ot unlike the paper electrophoretic pattern, t h e a g a r e l e c t r o p h e r o g r a m also s h o w s a p r o t e i n f r a c t i o n to b e p r e s e n t in v e r y h i g h c o n c e n t r a t i o n , w i t h i n t e r m e d i a t e m o b i l i t y , w h i c h is t e r m e d t h e p r i n c i p a l f r a c t i o n ( P F ) . T h e r e l a t i v e m o b i l i t y of t h i s f r a c t i o n £u t h e a g a r p l a t e ~ 4 t h a n ionic s t r e n g t h of 0.025 w a s qn~ = 0.39. W h e n t h e extrac~ of ~he c o r n e a l e p i t h e l i u m w a s v e r y f r e s h (less t h a n 1 h r old), o n l y a single, r a t h e r n a r r o w , p r i n c i p a l f r a c t i o n w a s fom~d (Fig. 1). E l e c t r o p h o r e s i s of a n older e x t r a c t (stored for 2 h r or longer), h o w e v e r , g e n e r a l l y s h o w e d two, or e v e n t h r e e , c o m p o n e n t s ~ i g r a t i n g r a t h e r close t o g e t h e r (:Fig. 2).
20.S
RF /
_
1"5
~
0"5 Relotive mobdity, mr
PF
0
Fro. i. A g a r elcctrophcrogram at / ' / 2 0.05 of the p r o t e i n s o f b o v i n e con]eal e p i t h e l i u m (fxesh ext r a c t ) . :Density our~-e a f t e r p r o t e i n s t a i n i n g . I~F, l~apid f r a c t i o n ; :PI,', p r i n c i p a l f r a c t i o n ; SF, slow f r a c t i o n . T h e b r e a k in t h e line r e p r e s e n t s t h e s l o t in ~vhich t h e e x t r a c t w a s p l a c e d .
O n t h e a n o d e side of t h e slot, w h e r e t h e p r o t e i n s o l u t i o n w a s a p p l i e d , t h e r e w a s a l w a y s a wide zone on t h c e l e c t r o p h e r o g r a m , w h e r e t h e p r o t e i n f r a c t i o n s h a d m i g r a t e d w i t h i n t e n s i v e s t r i a t i o n (STr¢). C o n s i d e r a b l e dii~erences w e r e n o t e d in t h e d i s t r i b u t i o n of t h e o t h e r f r a c t i o n s , d e p e n d i n g on t h e ionic streng%h u s e d in t h e e l e c t r o p h o r e t i c e x p e r i m e n t . C o m p a r i s o n of Figs. 1 a n d 2 p e r m i t s a n e s t i m a t e of t h e s e differences. E l e c t r o p h o r e s i s a t a n ionic s t r e n g t h of 0.05 r e v e a l e d a n a r r o w r a p i d f r a c t i o n (I~F) of h i g h c o n c e n t r a t i o n w i t h a r e l a t i v e m o b i l i t y of 0 "79 (:Fig. 1). T h i s f r a c t i o n w a s n o t f o u n d w h e n a n ionic s t r e n g t h of 0-025 w a s ~sed. A t l e a s t i n p a r t , t h i s r a p i d f r a c t i o n c o r r e s p o n d s to f r a c t i o n I
15151UNOEI,l]C'£1~OP~IO/~,ESIS
OF
COgNEAL
J~PI'I'H. ELIUM
199
obtained in p a p e r electrophoresis (:Fable 1). On the cathode side of t h e principal fraction, there were two i m p o r t a n t slow fractions (SF) a t 1"/2 0"05 and ordy one at
ST2 O.025). addition, all along t h e a g a r electropherogram, different protein bands of v e r y h)w concentravion (minor protein fractions) were observed. In
RF
PF
~- 0-5 _8
16
1
O-S
0
Relative mobility,mr Fro. 2. A g a r e l e e t r o p h c r o g r a m a t _P/2 0.025 o f t h e p r o t e i n s o f b o v i n e corneal e p i t h e l i u m (stored extract). Ply', P r i n c i p a l f r a c t i o n ; g F , r a p i d fra~'tion; SF, slow fraction. T h e b r e a k in t h e line r e p r e s e n t s tim slot in which t h e u x t r a c t was placed.
1 .~munoelectrophoretic analysis The results of immunoelectrophoresis are illustrated in ][~'igs. 3:and 4. The precipitation lines are discussed in t h e order of their a p p e a r a n c e on the a g a r plate. The first precipitation line which became visible in t h e course of t h e i m m u n o clectrophoretic e x p e r i m e n t corresponds to the principal fraction (PF). This line C RF
i
SFI
PF
AS
FIG. 3. Schematic (h'awing of the immunoeiec~rophoretic pattern ( F / 2 0.05).~A8, Trough containing antiserum; C, slot in which sample was placed; [RDF. rapid fraction; PF, principal fraction; SF l, slow fraction.
¢
SIR PF AS
C F r o . 4. S c h e m a t i c d r a w i n g o f t h e i m m u n o e l c c t r o p h o r e t i ¢ p a t t e r n (_r'/2 0.025). S T R , s t r i a t e d fraction; P F , p r i n c i p a l f r a c t i o n ; AS, t r o u g h c o n t a i n i n g a n t i s e r u m ; C, s l o t in which s a m p l e w a s p l a c e d . I n t h e upl)er p a r t o f t h e d i a g r a m , on t h e a n o d e side o f t h e slot, o n l y t h e c o m p l e x o f p r e c i p i t a t i o n lines corres p o n d i n g t o t h e s t r i a t e d f r a c t i o n s is shown.
a p p e a r e d a f t e r a few hours a n d r a p i d l y e x t e n d e d w i t h time. V/e h a v e a l r e a d y mentioned t h a t this principal fraction, w h e n t h e e x t r a c t of the corneal epithelium is n o t entirely fresh, generally shows two or three components. W h e n immlmoelectrophoresis is p e r f o r m e d with such an extract, we obtain a t first a v e r y a s y m m e t r i c a l
200
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p r e c i p i t a t i o n line or t w o s e p a r a t e lines a t t h e l e v e l of P F , w h i c h fuse r a p i d l y a n d s h o w a r e a c t i o n of c o m p l e t e i d e n t i t y ( p a t t e r n o f fusion). L a t e r on, a t l e a s t f o u r m o r e s h a r p p r e c i p i t a t i o n lines a p p e a r o n t h e a g a r p l a t e , w h i c h are well s e p a r a t e d f r o m e a c h o t h e r a n d s y m m e t r i c a l , so t h a t t h e r e c a n b e n o d o u b t t h a t t h e y are p r o d u c e d b y d i f f e r e n t a n t i g e n s . I t h a s b e e n i m p o s s i b l e t h u s far t o c o r r e l a t e all of t h e p r e c i p i t a t i o n lines w i t h t h e p r o t e i n f r a c t i o n s d e m o n s t r a t e d o n s i m p l e e l e c t r o p h o r e s i s . I n t h e p l a t e s w i t h 1"/2 0.05, i t w a s clear t h a t o n e p r e c i p i t a t i o n line c o r r e s p o n d s to t h e r a p i d f r a c t i o n ( R F ) a n d a n o t h e r to o n e of t h e slow f r a c t i o n s (SF~) (Figs. 1 a n d 3). PF
11 3
4 5 6 t
7 FIG. 5. Schematic drawing of the immunoeleetrophoretie patteru of proteins from the corneal epithelium of various mammals (/"/2 0-025). l, ox; 2, sheep; 3, horse; 4, pig; 5, dog; 6, cat; 7, man. ~PF,principal fraction. 2 0
0 3
B'
~
-,AS
3 1 2 :Fro. 6. Immunological analysis of the proteins from the eoraaea] epithelium by double-diffusion technique. A--Antiserum (AS) deposited in a central well; ]3--antiserum (AS) deposited in a linear trough. 1, bovine corneal epithelium; 2, porcine corneal epithelium; 3, bovine serum. F i n a l l y , w e f o u n d , a t t h e l e v e l of t h e s t r i a t e d f r a c t i o n s , a c o m p l e x o f p r e c i p i t a t i o n lines, s i t u a t e d v e r y close t o t h e a n t i g e n , w h i c h w e r e o f t e n v e r y l o n g a n d a s y m m e t r i c a l (Fig. 4, u p p e r part). I t h a s n o t y e t b e e n p o s s i b l e t o d e t e r m i n e w i t h c e r t a i n t y w h e t h e r t h e s e s t r i a t e d f r a c t i o n s ( S T R ) c o n t a i n e d one a n t i g e n or s e v e r a l a n t i g e n s . I t m u s t b e b o r n e in m i n d t h a t p r e c i p i t a t i o n lines m a y b e p r o d u c e d b y p r o t e i n f r a c t i o n s f r o m b o v i n e b l o o d , b e c a u s e it is i m p o s s i b l e t o o b t a i n f o r i m m u n i z a t i o n a
IMMUN0]~LECTI~OPHORESIS
OF CORNEAL
EPITHELIUM
201
large q u a n t i t y of corneal epithelium w i t h no trace of blood. Bovine haemoglobin p r o d u c e d no precipitation line in t h e presence of our antiserum. I m m u n o e l e c t r o phoresis of bovine blood serum, developed with our a n t i s e r u m to bovine corneal epithelium, a l w a y s p r o d u c e d a precipitation line a t the level of t h e ¢¢2-globulin and, a t least w i t h some of t h e antisera, a n o t h e r line a t t h e level of t h e ~,-globulin. W e are convinced t h a t none of t h e p r e c i p i t a t i o n lines shown in our figures corresponds with those p r o d u c e d b y t h e s e r u m proteins. This was ascertained b y a careful check of the mobilities a n d b y using a n t i s e r u m w h i c h h a d been absorbed with bovine serum, t h u s containing no antibodies a g a i n s t t h e bovine s e r u m - p r o t e i n fractions.
Comparative immunochemistry F o r comparison, immunoelectrophoresis was also applied to the proteins of the corneal epithelium of h u m a n s a n d of different animals, viz. sheep, horses, pigs, dogs a n d cats (Fig. 5). l~abbit a n t i s e r u m to bovine corneal epithelium was used in every case. I n e v e r y instance, a m o r e or less pronounced precipitation line was found a t the level of t h e protein fraction of h i g h e s t concentration, i.e. P F . The relative mobility of P F varied considerably in t h e different animal species examined. Only in sheep was a second precipitation line found. Using ~he double-diffusion technique, we were able to d e m o n s t r a t e t h a t t h e principal fraction of t h e corneal epithelium of the animals e x a m i n e d shows a reaction of p a r t i a l i d e n t i t y w i t h t h a t of t h e bovine corneal epithelium ( p a t t e r n of p a r t i a l intersection), lVigu~e 6 is a schematic d r a w i n g of a d o u b l e - ~ i o n a g a r p l a t e on which the proteins of porcine corneal epithelium were developed side b y side w i t h those of bovine corneal ep~thelinm. This reaction of p a r t i a l i d e n t i t y was also n o t e d w h e n h u m a n corneal epithelium was used.
4. D i s c u s s i o n
As has been observed with o t h e r tissue e x t r a c t s (Francois a n d l~abaey, 1959; Wieme, 1959a), a g a r microelectrophoresis ensures b e t t e r f r a e t i o n a t i o n of t h e soluble proteins of t h e corneal epithelium. A t a relatively high ionic strength, a p a t t e r n can be o b t a i n e d which shows some resemblance to t h a t o b t a i n e d b y p a p e r electrophoresis. This is n o t surprising, since p a p e r electrophoresis was carried out a t 1"/2 0-1. I n addition, however, several m i n o r fractions were f o u n d which are n o t revealed b y p a p e r electrophoresis. The significance of the large a m o u n t of protein which m i g r a t e s with intensive s t r i a t i o n is still u n k n o w n . These s t r i a t e d fractions are always m o s t i m p o r t a n t a f t e r electrophoresis of e x t r a c t s from y o u n g or active cells. This c o m p l e x i t y in the protein composition of t h e corneal epithelium is confirmed b y immunoelectrophoresis, which shows a t least eight different precipitation lines. I t m u s t also be pointed o u t t h a t n o t all of t h e p r o t e i n fractions t h a t can be seen a f t e r p r o t e i n staining of t h e electropherogram developed precipitation lines during the immunoelectrophoretic experiment. I t is e v i d e n t t h a t all of these precipitation lines correspond to tissue proteins. All of t h e m were also f o u n d w h e n a n t i s e r u m absorbed with bovine s e r u m was used. This conf~rms t h e e x p e r i m e n t s of K a w e r a u a n d O t t (1961), who detected o n l y one precipitation line (corresponding to s e r u m albumin) a f t e r immunoelectrophoresis of corneal epitheli~lm developed w i t h antisemlm to s e r u m protein.~. The serum proteins t h u s form only a small p a r t of t h e soluble proteins of t h e corneal epithelium.
202
JULES FRANCOIS AND MARCEL R A B A E Y
:[mmunoelectrophot'etic investigation has made it possible to obtain new data on the principal protein fraction of the corneal epithelium. The facility of demonstrating this P F in different m a m m a l s with "antibovinc-corneal-epithelium" antiserum, as well as the pattern of partial intersection always obtained with bovine PF, suggest that this protein possesses a considerable degree of organ specificity.
REFERENCES Burtin, P. (1960). In Analyse Immuno~lectrophor~tlque, ed. by P. Grabar and P. Burtin, p. 84. Masson, Paris. Franqois, J. and ]~abaey, M. (1954). Bull. Soc. belge Ophtal. 108, 641. Franqois, J. and Rabaey, M. (1956). Ann. Oculist., Paris 189, 445. Fran~,:ois, J. and Rabacy, M. (1959). A.M.A. Arch. Ophthal. 61,531. Francois, J., Rabaey, M. and Weinstein, R. (1961). Ann. Oculist., Paris 194, 589. Guidry, M. A., Kelly, J. B. and Allen, J. ][. (1956). Prec. Soc. exp. Biol. N . Y . 92, 469. Kawerau, E. and Ott, H. (1961). Exp. Eye JCes. 1, 137. Miinich, W. and Oswald, A. (1955). v. Oracles Arch. Ophthal. 156, 547. Ouehterlony, C). (1958). Progr. Allergy 5, 1. Rabaey, M. and Verriest, G. (1957). Ann. Soc. zool. Belge 88, 373. Rabaey, 1~I. (1959). Over de Eiwitsamenstelling der Ooglens. Thesis, University of" Ghent, Belgium. Seheidegger, J. J. (1955). Int. Arch. Allergy, 7, 103. St~invorth, E. (1958). v. Graefes Arch. OphthaI. 160, 271. Steinvorth, E. (1959a). v. Oraefes Arch. Ophthal. 160, 540. Steinvorth, E. (1959b). v. Graefes Arch. Ophthal. 160, 588. Steinvorth, E. (1960a). v. Graefes Arch. Ophtluzl. 161,466. Stcinvorth, E. (1960b). v. Graefe,~ Arch. Ophtha?. 162, 28. Takaku, I. (1957). Acts Soc. Ophthal. Jap. 61, r~2~'. Wieme, R. J. (1959a). Studies on Agar Gel :b"~'ctrophoresis. Thesis, Univel~ity of Ghent, Belgium. Wieme, 1~. J. (1959b). Clin. Chim. Acts 4, 317.
lmmunoelectrophoresis of the Proteins of the Corneal Epithelium J U L E S .~ [
O'phthalmolog'icat Clinic, of the University of Ghe.nt, Belgium (Receiced 27 Febr~lary 1962 and in ret,'isedJbrm 12 Ju~e 1962) The protehls of the corne~l epithelium are separated into a greater number of tractions by high-tension agar electrophoresis than by paper electrophoresis. The distribut i o n o f t h e d i f f e r e n t fr,~ctions is g r e a t l y i n f l u e n c e d b y t h e i o n i c s t r e n g t h o f t h e b u f f e r s o l u t i o n . A l t h o u g h in a g a r e l e e t r o p h o r e s i s a l a r g e r n u m b e r o f f r a c t i o n s a r e f o u n d , t h e r e is, n e v e ~ h e l e s s , a g r e a t s i m i l a r i t y b e t w e e n t h e p a t t e r n o b t a i n e d a f t e r p a t ) e r e l e c t r o phoresis and that obtained after agar electrophoresis at relatively high ionic strength. T h e e x i s t e n c e o f a g r e a t e r n u m b e r o f f r a c t i o n s is c o n f i r m e d b y i m m u n o e l e c t r o p h o r e s i s , which reveals at least eight different precipitation liues corresponding to different tissue proteins. T h e f r a c t i o n p r e s e n t i n h i g h e s t c o n c e n t r a t i o n , w h i c h wo h a v e c a l l e d t h e p r i n c i p a l f r a c t i o n , is f o u n d n o t o n l y i n h u m a n s , b u t a l s o in s e v e r a l o t h e r m a m m a l s . I n v i e w o f t h e f a c t t h a t , in all c a s e s , t h e c o r r e s p o n d i n g p r e c i p i t a t i o n l i n e a p p e a r e d w h e n a n t i s e r u m to bovine corneal epithelium was used, it would seem that the principal fraction has a v e r y d e f i n i t e o r g a n s p e c i f i c i t y . T h i s f r a c t i o n h a s p o o r s t a b i l i t y in s o l u t i o n . I t r a p i d l y decomposes into two or three components, which are separated during electrophoresis in agar, where their mobiHties are slightly different.
1. Introduction
Several authors have studied the water-soluble proteins of the corneal epithelium b y paper electrophoresis (Franpois and l%abaey, 195~, 1956; Miinich and Oswald, 1955; Takak~u, 1957; Steinvorth, 1958, 1959a,b, 1960a,b). Other authors have studied the soluble proteins of the entire cornea, h~cluding the epithelium and the parenchyana, TA]~LE I
Soluble prolein f~'action.s' of bovine corneal e'oithelium after separation by paper electro phorcsis Pi-otcin freer ion 1
l"ranqois a n d R a b a e y ( 1954 )
)litnich a n d Oswald ( 1 !)55)
Steinvorth (1960a)
l(~awerau a n d O t t ( 1901 )
12
3-63
19"3
ll'0
]I ]] I
(13 O
20-93 59-55
60"5 12 "0
05-0 16 "3
j \r
7
15-80
8-2
3.7
4.0 ~:a]tles
aT'~,
o., ,.o total lwoteins.
e.g. Guidry, Kelly and Allen (1956), who resorted to the determination of the sedi m e n t a t i o n constants after ultracentrifugation. Recently, l ( a w e r a u and Oft (1961) m a d e a st u d y of the soluble proteins of the three structural layers of the cornea, the epithelium, the substantia propria and the endothelium, also by paper eleetrophoresis. Most of these investigators have distinguished av least four soluble proteins in the paper electrophoretic p a t t e r n of the bovine corneal epithelium, b u t t h e y have reported different figures for the distribution of these fractions, as shown in Table I. Fraction III, which shows P~kS-positivity on paper electrophoresis (Franpois and 196
1MM. I J N O I g L E Q ' I Y I . I O P I t O I I , I g S I N O F C O I / N I ~ A I ,
EPI'I?I~I']I,1UM
197
I~abaey, 1954, ] 956), 1)robably c o n t a i n s g l y c o g e n in c o m p l e x w i t h proteiJls (Frangois, i~almey mxd W e i n s t e i n , 1_961). T h e s a m e a u t h o r s also f o n n d t h a t on t h e p a p e r electrop h e r o g r a m ~ o n l y ti'aetion IJ s t a i n e d s l i g h t l y w i t h S u d a n 131acl¢ 13, s u g g e s t i n g t h e presence of o n l y a small q u a r t t i t y of lipoproteiJm (FrangoLs a~ld ]Labaey, 1954, 1956). S t e i n v o r t h (1958), h o w e v e r , f o u n d t w o l i p o p r o t e i n tYactions w h i c h , air.hough iJ~ different p r o p o r t i o n , b o t h s t a i n e d p o s i t i v e l y for p h o s p h o l i p o p r o t e h l s . The agar electrophoretic and imarmnoeleetrophoretic studies presented in this p a p e r were c a r r i e d o u t for t h e p u r p o s e of c h a r a c t e r i z i n g m o r e a c c u r a t e l y t h e d i f f e r e n t w a t e r - s o l u b l e p r o t e i n s of t h e c o r n e a l e p i t h e l i u m , t h e n a t u r e a n d f u n c t i o n of wlfich are still a l m o s t c o m p l e t e l y u n k n o w n . 2. M a t e r i a l s and M e t h o d s
The bovine a n d other a n i m a l eyes were o b t a i n e d within 10 min after the d e a t h of t h e animal. The h u m a n eye was o b t a i n e d a t operation, the ey.e being removed for uveal melanoma. The corneal and c o n j u n c t i v a l surfaces of the eyes were wiped w i t h a cloth soaked in l~inger's solution to remove t h e blood. The epithelial cells of the cornea were isolated w i t h a Graefe knife b y a t e c h n i q u e w h i c h has been previously described (lq'ran~ois and R a b a e y , 1954, ]956). I n general, it is relatively easy to separate the entire epithelium from t h e u n d e r l y i n g stroma. :For i m m u n i z a t i o n , t h e bovine corneal epithelium was homogenized in :Ringer's solution, using 100 mg of fresh tissue per ml of solution. The suspension was t h e n centrifuged a t 20,000 × g for 20 rain. One milliliter of the s u p e r n a t a n t , which c o n t a i n e d a b o u t 1 % proteins (protein concent r a t i o n d e t e r m i n e d b y t h e B i u r e t method), was injected once each week for 6-8 weeks s u b c u t a n e o u s l y into r a b b i t s weighing a b o u t 2 kg. The serum of t h e i n j e c t e d animals was removed 2 weeks after the l a s t injection. A certain a m o u n t of this a n t i s e r u m was absorbed with the n o r m a l bovine serum, as described b y ]3urtin (1960). U p to 0"1 ml of bovine serum was a d d e d to 1 ml of antiserum. D u r i n g immunodiffusion or immunoelectrophoresis, no p r e c i p i t a t i o n lines occurred, w h e n t h e bovine serum was developed w i t h t h e absorbed antiserum. :For electrophoresis, the corneal epithelium was homogenized in R i n g e r ' s solution or in distilled w a t e r using 200 m g of fresh tissue per m | of solution. After c e n t r i f u g a t i o n as indicated above, the clear s u p e r n a t a n t , which c o n t a i n e d a b o u t 2 % protein, was used for electrophoresis a n d immmmelectrophoresis w i t h o u t dialysis. On storage, a g r a d u a l clouding was observed in the solution and, after a few hours, a slight precipitate was formed. Electrophoresis was carried o u t in agar on microscopic slides a t 4°C a n d a t h i g h tension (20 V/cm) (Rabaey, 1959; Wieme, 1959a,b). C o n s t a n t t e m p e r a t u r e was achieved b y means of an a u t o m a t i c cooling s y s t e m in which p e t r o l e u m ether (b.p. 30°-50°C) was used in t h e electrophoresis t a n k as i m m e r s i n g fluid. As s u p p o r t i n g medium, either Difco Special Ag~r Noble* (1~/o in veronal buffer, p H 8-~, _P/2 0.05) or Difco Bacto-Agar* (1~/o in veronal buffer, ptff 8-4, I / 2 0-025) was used. On completion of electrophoresis (25 rain), the a g a r plates were fixed in 6 5 % e t h y l alcohol c o n t a i n i n g 5~/o acetic acid (w/w) for 1-24- hr. After d r y i n g u n d e r filter paper, t h e plates were finally stained a n d scanned. The s t a i n i n g solution was composed of 0"5 g of amido black 10B, 40 ml of normal acetic acid, 10 m l of normal sodium acetate, 10 m l of glycerin a n d 40 ml of distilled water. ~tfter s t a i n i n g for 30 rain, t h e agar plates are differentiated in 2 % acetic acid. Scanning was performed a t 546 m~x in a modified :Eppendorf p h o t o m e t e r (\Vieme, 1959a). The density m~rves were o b t a i n e d b y p l o t t i n g the absorbance A of the s t a i n e d protein fractions a g a i n s t the relative m o b i l i t y m~. The relative m o b i l i t y ~ of the different fractions was d e t e r m i n e d as described earlier ( R a b a e y a n d Verriest, 1957), using d e x t r a n (mr=O) a n d h u m a n serum a l b u m i n ( m r s 1 ) as t e s t substances. * Dffco : L a b o r a t o r i e s , D e t r o i t , Mich.
198
,JULES
FRANq~OIS
AND
MAgC.~]L
I~ABAEY
I m m u n o e l e c t r o p h o r c s i s was also carried out on microscopic slides as suggested by the Scheidcgger technique (1955). A trough, 1 m m wide a n d 6-7 cm long, was m~,.de in the middle of the agar plate, r u n n i n g parallel to the long side of the slide. The e x t r a c t of the corneal epithelium was deposited in the centre of the plate, a b o u t 3 m m from both sides of t h e trough in a slot 1-2 m m long. Electrophoresis was t h e n carried o u t as described above, after which t h e trough was filled with a n t i s e r u m and the plates were allowed to d e v e l o p for 24 hr. The best results were obtained with u n d i l u t e d antiserum. l m m u n o d i f f u s i o n tests (double-diflhsion t e c h n i q u e according to Ouchterlony, 1958) were carried out on micro-plates usi~g two different templates (Fig. 6). T h e first t e m p l a t e consists of a central well, filled w i t h antiserum, stu-rounded b y several others filled with different antigens; in t h e second t e m p l a t e the different antigen wells are situated along a trench-like well filled with antiserum. 3. R e s u l t s
Agar electro~horetic analysis
T h e d e n s i t y ctu'ves of a g a r e l e c t r o p h e r o g r a m s o b t a i n e d w i t h proteh~s f r o m b o v i n e c o r n e a l e p i t h e l i l m l a t t w o d i f f e r e n t ionic s t r e n g t h s are s h o ~ m in :Fig. 1 (Difco Special A g a r ~ o b l e , 1"/2 0.05) a n d Fig. 2 (Difco B a c t o - A g a r , /"/2 0-025). I t is o b v i o u s t h a t t h e p a t t e r n s o b t a i n e d w i t h t h i s m e t h o d are m o r e c o m p l e x th,~n those obtained b y paper elcctrophoresis. N ot unlike the paper electrophoretic pattern, t h e a g a r e l e c t r o p h e r o g r a m also s h o w s a p r o t e i n f r a c t i o n to b e p r e s e n t in v e r y h i g h c o n c e n t r a t i o n , w i t h i n t e r m e d i a t e m o b i l i t y , w h i c h is t e r m e d t h e p r i n c i p a l f r a c t i o n ( P F ) . T h e r e l a t i v e m o b i l i t y of t h i s f r a c t i o n £u t h e a g a r p l a t e ~ 4 t h a n ionic s t r e n g t h of 0.025 w a s qn~ = 0.39. W h e n t h e extrac~ of ~he c o r n e a l e p i t h e l i u m w a s v e r y f r e s h (less t h a n 1 h r old), o n l y a single, r a t h e r n a r r o w , p r i n c i p a l f r a c t i o n w a s fom~d (Fig. 1). E l e c t r o p h o r e s i s of a n older e x t r a c t (stored for 2 h r or longer), h o w e v e r , g e n e r a l l y s h o w e d two, or e v e n t h r e e , c o m p o n e n t s ~ i g r a t i n g r a t h e r close t o g e t h e r (:Fig. 2).
20.S
RF /
_
1"5
~
0"5 Relotive mobdity, mr
PF
0
Fro. i. A g a r elcctrophcrogram at / ' / 2 0.05 of the p r o t e i n s o f b o v i n e con]eal e p i t h e l i u m (fxesh ext r a c t ) . :Density our~-e a f t e r p r o t e i n s t a i n i n g . I~F, l~apid f r a c t i o n ; :PI,', p r i n c i p a l f r a c t i o n ; SF, slow f r a c t i o n . T h e b r e a k in t h e line r e p r e s e n t s t h e s l o t in ~vhich t h e e x t r a c t w a s p l a c e d .
O n t h e a n o d e side of t h e slot, w h e r e t h e p r o t e i n s o l u t i o n w a s a p p l i e d , t h e r e w a s a l w a y s a wide zone on t h c e l e c t r o p h e r o g r a m , w h e r e t h e p r o t e i n f r a c t i o n s h a d m i g r a t e d w i t h i n t e n s i v e s t r i a t i o n (STr¢). C o n s i d e r a b l e dii~erences w e r e n o t e d in t h e d i s t r i b u t i o n of t h e o t h e r f r a c t i o n s , d e p e n d i n g on t h e ionic streng%h u s e d in t h e e l e c t r o p h o r e t i c e x p e r i m e n t . C o m p a r i s o n of Figs. 1 a n d 2 p e r m i t s a n e s t i m a t e of t h e s e differences. E l e c t r o p h o r e s i s a t a n ionic s t r e n g t h of 0.05 r e v e a l e d a n a r r o w r a p i d f r a c t i o n (I~F) of h i g h c o n c e n t r a t i o n w i t h a r e l a t i v e m o b i l i t y of 0 "79 (:Fig. 1). T h i s f r a c t i o n w a s n o t f o u n d w h e n a n ionic s t r e n g t h of 0-025 w a s ~sed. A t l e a s t i n p a r t , t h i s r a p i d f r a c t i o n c o r r e s p o n d s to f r a c t i o n I
15151UNOEI,l]C'£1~OP~IO/~,ESIS
OF
COgNEAL
J~PI'I'H. ELIUM
199
obtained in p a p e r electrophoresis (:Fable 1). On the cathode side of t h e principal fraction, there were two i m p o r t a n t slow fractions (SF) a t 1"/2 0"05 and ordy one at
ST2 O.025). addition, all along t h e a g a r electropherogram, different protein bands of v e r y h)w concentravion (minor protein fractions) were observed. In
RF
PF
~- 0-5 _8
16
1
O-S
0
Relative mobility,mr Fro. 2. A g a r e l e e t r o p h c r o g r a m a t _P/2 0.025 o f t h e p r o t e i n s o f b o v i n e corneal e p i t h e l i u m (stored extract). Ply', P r i n c i p a l f r a c t i o n ; g F , r a p i d fra~'tion; SF, slow fraction. T h e b r e a k in t h e line r e p r e s e n t s tim slot in which t h e u x t r a c t was placed.
1 .~munoelectrophoretic analysis The results of immunoelectrophoresis are illustrated in ][~'igs. 3:and 4. The precipitation lines are discussed in t h e order of their a p p e a r a n c e on the a g a r plate. The first precipitation line which became visible in t h e course of t h e i m m u n o clectrophoretic e x p e r i m e n t corresponds to the principal fraction (PF). This line C RF
i
SFI
PF
AS
FIG. 3. Schematic (h'awing of the immunoeiec~rophoretic pattern ( F / 2 0.05).~A8, Trough containing antiserum; C, slot in which sample was placed; [RDF. rapid fraction; PF, principal fraction; SF l, slow fraction.
¢
SIR PF AS
C F r o . 4. S c h e m a t i c d r a w i n g o f t h e i m m u n o e l c c t r o p h o r e t i ¢ p a t t e r n (_r'/2 0.025). S T R , s t r i a t e d fraction; P F , p r i n c i p a l f r a c t i o n ; AS, t r o u g h c o n t a i n i n g a n t i s e r u m ; C, s l o t in which s a m p l e w a s p l a c e d . I n t h e upl)er p a r t o f t h e d i a g r a m , on t h e a n o d e side o f t h e slot, o n l y t h e c o m p l e x o f p r e c i p i t a t i o n lines corres p o n d i n g t o t h e s t r i a t e d f r a c t i o n s is shown.
a p p e a r e d a f t e r a few hours a n d r a p i d l y e x t e n d e d w i t h time. V/e h a v e a l r e a d y mentioned t h a t this principal fraction, w h e n t h e e x t r a c t of the corneal epithelium is n o t entirely fresh, generally shows two or three components. W h e n immlmoelectrophoresis is p e r f o r m e d with such an extract, we obtain a t first a v e r y a s y m m e t r i c a l
200
J U L E S FI:tANQOIS AND MAI~CEL I~A:BAEY
p r e c i p i t a t i o n line or t w o s e p a r a t e lines a t t h e l e v e l of P F , w h i c h fuse r a p i d l y a n d s h o w a r e a c t i o n of c o m p l e t e i d e n t i t y ( p a t t e r n o f fusion). L a t e r on, a t l e a s t f o u r m o r e s h a r p p r e c i p i t a t i o n lines a p p e a r o n t h e a g a r p l a t e , w h i c h are well s e p a r a t e d f r o m e a c h o t h e r a n d s y m m e t r i c a l , so t h a t t h e r e c a n b e n o d o u b t t h a t t h e y are p r o d u c e d b y d i f f e r e n t a n t i g e n s . I t h a s b e e n i m p o s s i b l e t h u s far t o c o r r e l a t e all of t h e p r e c i p i t a t i o n lines w i t h t h e p r o t e i n f r a c t i o n s d e m o n s t r a t e d o n s i m p l e e l e c t r o p h o r e s i s . I n t h e p l a t e s w i t h 1"/2 0.05, i t w a s clear t h a t o n e p r e c i p i t a t i o n line c o r r e s p o n d s to t h e r a p i d f r a c t i o n ( R F ) a n d a n o t h e r to o n e of t h e slow f r a c t i o n s (SF~) (Figs. 1 a n d 3). PF
11 3
4 5 6 t
7 FIG. 5. Schematic drawing of the immunoeleetrophoretie patteru of proteins from the corneal epithelium of various mammals (/"/2 0-025). l, ox; 2, sheep; 3, horse; 4, pig; 5, dog; 6, cat; 7, man. ~PF,principal fraction. 2 0
0 3
B'
~
-,AS
3 1 2 :Fro. 6. Immunological analysis of the proteins from the eoraaea] epithelium by double-diffusion technique. A--Antiserum (AS) deposited in a central well; ]3--antiserum (AS) deposited in a linear trough. 1, bovine corneal epithelium; 2, porcine corneal epithelium; 3, bovine serum. F i n a l l y , w e f o u n d , a t t h e l e v e l of t h e s t r i a t e d f r a c t i o n s , a c o m p l e x o f p r e c i p i t a t i o n lines, s i t u a t e d v e r y close t o t h e a n t i g e n , w h i c h w e r e o f t e n v e r y l o n g a n d a s y m m e t r i c a l (Fig. 4, u p p e r part). I t h a s n o t y e t b e e n p o s s i b l e t o d e t e r m i n e w i t h c e r t a i n t y w h e t h e r t h e s e s t r i a t e d f r a c t i o n s ( S T R ) c o n t a i n e d one a n t i g e n or s e v e r a l a n t i g e n s . I t m u s t b e b o r n e in m i n d t h a t p r e c i p i t a t i o n lines m a y b e p r o d u c e d b y p r o t e i n f r a c t i o n s f r o m b o v i n e b l o o d , b e c a u s e it is i m p o s s i b l e t o o b t a i n f o r i m m u n i z a t i o n a
IMMUN0]~LECTI~OPHORESIS
OF CORNEAL
EPITHELIUM
201
large q u a n t i t y of corneal epithelium w i t h no trace of blood. Bovine haemoglobin p r o d u c e d no precipitation line in t h e presence of our antiserum. I m m u n o e l e c t r o phoresis of bovine blood serum, developed with our a n t i s e r u m to bovine corneal epithelium, a l w a y s p r o d u c e d a precipitation line a t the level of t h e ¢¢2-globulin and, a t least w i t h some of t h e antisera, a n o t h e r line a t t h e level of t h e ~,-globulin. W e are convinced t h a t none of t h e p r e c i p i t a t i o n lines shown in our figures corresponds with those p r o d u c e d b y t h e s e r u m proteins. This was ascertained b y a careful check of the mobilities a n d b y using a n t i s e r u m w h i c h h a d been absorbed with bovine serum, t h u s containing no antibodies a g a i n s t t h e bovine s e r u m - p r o t e i n fractions.
Comparative immunochemistry F o r comparison, immunoelectrophoresis was also applied to the proteins of the corneal epithelium of h u m a n s a n d of different animals, viz. sheep, horses, pigs, dogs a n d cats (Fig. 5). l~abbit a n t i s e r u m to bovine corneal epithelium was used in every case. I n e v e r y instance, a m o r e or less pronounced precipitation line was found a t the level of t h e protein fraction of h i g h e s t concentration, i.e. P F . The relative mobility of P F varied considerably in t h e different animal species examined. Only in sheep was a second precipitation line found. Using ~he double-diffusion technique, we were able to d e m o n s t r a t e t h a t t h e principal fraction of t h e corneal epithelium of the animals e x a m i n e d shows a reaction of p a r t i a l i d e n t i t y w i t h t h a t of t h e bovine corneal epithelium ( p a t t e r n of p a r t i a l intersection), lVigu~e 6 is a schematic d r a w i n g of a d o u b l e - ~ i o n a g a r p l a t e on which the proteins of porcine corneal epithelium were developed side b y side w i t h those of bovine corneal ep~thelinm. This reaction of p a r t i a l i d e n t i t y was also n o t e d w h e n h u m a n corneal epithelium was used.
4. D i s c u s s i o n
As has been observed with o t h e r tissue e x t r a c t s (Francois a n d l~abaey, 1959; Wieme, 1959a), a g a r microelectrophoresis ensures b e t t e r f r a e t i o n a t i o n of t h e soluble proteins of t h e corneal epithelium. A t a relatively high ionic strength, a p a t t e r n can be o b t a i n e d which shows some resemblance to t h a t o b t a i n e d b y p a p e r electrophoresis. This is n o t surprising, since p a p e r electrophoresis was carried out a t 1"/2 0-1. I n addition, however, several m i n o r fractions were f o u n d which are n o t revealed b y p a p e r electrophoresis. The significance of the large a m o u n t of protein which m i g r a t e s with intensive s t r i a t i o n is still u n k n o w n . These s t r i a t e d fractions are always m o s t i m p o r t a n t a f t e r electrophoresis of e x t r a c t s from y o u n g or active cells. This c o m p l e x i t y in the protein composition of t h e corneal epithelium is confirmed b y immunoelectrophoresis, which shows a t least eight different precipitation lines. I t m u s t also be pointed o u t t h a t n o t all of t h e p r o t e i n fractions t h a t can be seen a f t e r p r o t e i n staining of t h e electropherogram developed precipitation lines during the immunoelectrophoretic experiment. I t is e v i d e n t t h a t all of these precipitation lines correspond to tissue proteins. All of t h e m were also f o u n d w h e n a n t i s e r u m absorbed with bovine s e r u m was used. This conf~rms t h e e x p e r i m e n t s of K a w e r a u a n d O t t (1961), who detected o n l y one precipitation line (corresponding to s e r u m albumin) a f t e r immunoelectrophoresis of corneal epitheli~lm developed w i t h antisemlm to s e r u m protein.~. The serum proteins t h u s form only a small p a r t of t h e soluble proteins of t h e corneal epithelium.
202
JULES FRANCOIS AND MARCEL R A B A E Y
:[mmunoelectrophot'etic investigation has made it possible to obtain new data on the principal protein fraction of the corneal epithelium. The facility of demonstrating this P F in different m a m m a l s with "antibovinc-corneal-epithelium" antiserum, as well as the pattern of partial intersection always obtained with bovine PF, suggest that this protein possesses a considerable degree of organ specificity.
REFERENCES Burtin, P. (1960). In Analyse Immuno~lectrophor~tlque, ed. by P. Grabar and P. Burtin, p. 84. Masson, Paris. Franqois, J. and ]~abaey, M. (1954). Bull. Soc. belge Ophtal. 108, 641. Franqois, J. and Rabaey, M. (1956). Ann. Oculist., Paris 189, 445. Fran~,:ois, J. and Rabacy, M. (1959). A.M.A. Arch. Ophthal. 61,531. Francois, J., Rabaey, M. and Weinstein, R. (1961). Ann. Oculist., Paris 194, 589. Guidry, M. A., Kelly, J. B. and Allen, J. ][. (1956). Prec. Soc. exp. Biol. N . Y . 92, 469. Kawerau, E. and Ott, H. (1961). Exp. Eye JCes. 1, 137. Miinich, W. and Oswald, A. (1955). v. Oracles Arch. Ophthal. 156, 547. Ouehterlony, C). (1958). Progr. Allergy 5, 1. Rabaey, M. and Verriest, G. (1957). Ann. Soc. zool. Belge 88, 373. Rabaey, 1~I. (1959). Over de Eiwitsamenstelling der Ooglens. Thesis, University of" Ghent, Belgium. Seheidegger, J. J. (1955). Int. Arch. Allergy, 7, 103. St~invorth, E. (1958). v. Graefes Arch. OphthaI. 160, 271. Steinvorth, E. (1959a). v. Oraefes Arch. Ophthal. 160, 540. Steinvorth, E. (1959b). v. Graefes Arch. Ophthal. 160, 588. Steinvorth, E. (1960a). v. Graefes Arch. Ophtluzl. 161,466. Stcinvorth, E. (1960b). v. Graefe,~ Arch. Ophtha?. 162, 28. Takaku, I. (1957). Acts Soc. Ophthal. Jap. 61, r~2~'. Wieme, R. J. (1959a). Studies on Agar Gel :b"~'ctrophoresis. Thesis, Univel~ity of Ghent, Belgium. Wieme, 1~. J. (1959b). Clin. Chim. Acts 4, 317.